RESEARCH ARTICLE
RhoB Acts as a Tumor Suppressor That Inhibits Malignancy of Clear Cell Renal Cell Carcinoma Weihao Chen1,2☯‡, Shaoxi Niu1☯‡, Xin Ma1, Peng Zhang1, Yu Gao1, Yang Fan1, Haigang Pang1, Huijie Gong1, Donglai Shen1, Liangyou Gu1, Yu Zhang1, Xu Zhang1* 1 The State Key Laboratory of Kidney Diseases, Department of Urology, Military Postgraduate Medical College, Chinese People's Liberation Army General Hospital, Beijing, People's Republic of China, 2 Department of Urology, General Hospital of the Navy, Beijing, China
a11111
☯ These authors contributed equally to this work. ‡ These authors are joint senior authors on this work. * [email protected]
Abstract OPEN ACCESS Citation: Chen W, Niu S, Ma X, Zhang P, Gao Y, Fan Y, et al. (2016) RhoB Acts as a Tumor Suppressor That Inhibits Malignancy of Clear Cell Renal Cell Carcinoma. PLoS ONE 11(7): e0157599. doi:10.1371/journal.pone.0157599 Editor: Hiromu Suzuki, Sapporo Medical University, JAPAN Received: January 23, 2016 Accepted: May 31, 2016 Published: July 6, 2016 Copyright: © 2016 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National High Technology Research and Development Program of China (2014AA020607) to XM and NGH Innovation Fostering Fund (CXPY201423). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
This study aims to investigate the biological role of RhoB in clear cell renal cell carcinoma (ccRCC). The expression of RhoB was examined in specimens of patients and cell lines by Western blot and Immunohistochemistry. The correlation between RhoB expression and clinicopathologic variables was also analyzed. The effects of RhoB on cell proliferation, cell cycle, cell apoptosis, and invasion/migration were detected by over-expression and knockdown of RhoB level in ccRCC cells via plasmids and RNAi. The results showed that RhoB was low-expressed in ccRCC surgical specimens and cell lines compared with adjacent normal renal tissues and normal human renal proximal tubular epithelial cell lines (HKC), and its protein expression level was significantly associated with the tumor pathologic parameter embracing tumor size(P = 0.0157), pT stage(P = 0.0035), TNM stage(P = 0.0024) and Fuhrman tumor grade(P = 0.0008). Further, over-expression of RhoB remarkably inhibited the cancer cell proliferation, colony formation and promoted cancer cell apoptosis, and aslo reduced the invasion and migration ability of ccRCC cells. Interestingly, up-regulation of RhoB could induce cell cycle arrest in G2/M phase and led to cell cycle regulators(CyclineB1,CDK1) and pro-apoptotic protein(casp3,casp9) aberrant expression. Moreover, knockdown of RhoB in HKC cells promoted cell proliferation and migration. Taken together, our study indicates that RhoB expression is decreased in ccRCC carcinogenesis and progression. Up-regulation of RhoB significantly inhibits ccRCC cell malignant phenotype. These findings show that RhoB may play a tumor suppressive role in ccRCC cells, raising its potential value in futural therapeutic target for the patients of ccRCC.
Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
Introduction Clear cell renal cell carcinoma (ccRCC) originates from proximal tubule cells, and is one of the most common histological subtypes of renal cell carcinomas. ccRCC is the second leading cause of death among all types of urologic cancers[1, 2]. In fact, approximately 25% to 30% of the patients with ccRCC present metastasis at the time of diagnosis, and overall survival is usually very poor in the follow-up period[3]. Unfortunately, ccRCC is resistant to conventional cytotoxic agents, in addition to surgery[4].Although the new targeted therapies have produced dramatic clinical effects for the treatment of metastatic renal-cell carcinoma (RCC), such targeted therapies remain unsatisfactory because some patients are resistant to therapy [5].Thus, further studies are necessary to investigate the tumorigenesis and progression of ccRCC and to explore new therapeutic targets to improve the efficiency of ccRCC treatment. RhoB is a member of the Rho family of small GTPases, which regulates actin stress fibers, cytoskeletal actin organization and vesicle transport, in cancer cells, RhoB also modulates proliferation, survival, invasion and angiogenic capacity[6]. Furthermore, RhoB may act as a tumor suppressor in growth control and transformation. RhoB is not mutated in various cancers, but its altered expression and activity are possibly critical to cancer progression and therapeutic responses therapeutic responses[7, 8]. Loss of RhoB expression has been reported in head and neck cancer, lung cancer and gastric cancer[9–11]. RhoB gene knockout in mouse increases the frequency of chemically induced neoplastic transformation[12]. Overexpression of RhoB in human tumor cells results in inhibition of signal transduction pathways involved in oncogenesis and tumor survival, as well as apoptosis[13]. Studies have revealed the putative tumor-suppressive effect of RhoB in human tumor, however, to the best of our knowledge, the function of RhoB in ccRCC remains unclear. In the present study, the relative expression levels of RhoB in ccRCC cell lines and patient specimens were investigated by Western blot and immunohistochemistry. The correlation between RhoB expression and clinicopathological parameters of patients with ccRCC was also analyzed. The biological effects of overexpression and low-expression of RhoB on the malignant phenotypes of ccRCC cell A498,786-O and Caki-1 or normal HKC cells were further examined.
Materials and Methods Ethics Statement All patients authorized the Written Informed Consent. This study was approved by the Protection of Human Subjects Committee, Chinese People’s Liberation Army (PLA) General Hospital.
Cell culture and reagents Human renal proximal tubular epithelial cell line HKC and HK2, and the renal cancer cell lines, including A498, 786-O, 769-P and Caki-1, Caki-2 were preserved in our laboratory. The cells were maintained in DMEM or RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 units/ml of penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C.
Patients and tissue samples All ccRCC cases diagnosed clinically and histopathologically were obtained from Chinese People’s Liberation Army General Hospital (Beijing, China) in 2011. The study were approved by the Chinese People’s Liberation Army General Hospital’s Protection of Human Subjects Committee and the informed consent was obtained from all patients. After resection was performed,
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
specimens were promptly frozen in liquid nitrogen and stored at -80°C until use. In addition, parts of each sample were fixed in formalin, embedded in paraffin and stored in our laboratory. Inclusion criterion included: Patients who received radical nephrectomy in our hospital, the pathologic diagnosis was ccRCC, both the tumor specimens and renal tissues were available after surgery. Exclusion criterion were as follows:patients receiving chemotherapy or radiotherapy before surgery, with multiple renal tumors or distant metastasis.The following clinicopathologic information was collected for each patient,including age, sex, tumor size, pT status and TNM stage.
Protein extraction and Western-blot analysis Whole cell lysate was extracted from each cell line and surgical specimens in RIPA Lysis Buffer (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Protein concentration was measured using BCA Protein Quantitative Kit (Applygen Technologies). Protein (30μg) from each sample was separated in 8% or 12% SDS–PAGE, and transferred to polyvinylidene fluoride membranes (PVDF membranes, Millipore). The PVDF membranes were blocked with 5% low fat milk and then probed with the primary antibody RhoB(1:1000, Proteintech), CyclineB1(1:1000; Immunoway), CDK1(1:1000; Immunoway), cleaved casp9 (1:1000, Epitomics), and cleaved casp3(1:1000, Affinity BioReagents). β-actin(1:1000, ZSGB-BIO) was used as a loading control. Secondary anti-mouse or anti-rabbit antibodies were used at 1:3000 dilutions and the reaction was visualized using an enhanced chemiluminescent Kit (Thermo, IL).
Immunohistochemistry analysis The paraffin sections (4 μm in thickness) were deparaffinized in xylene solvent and rehydrated through a graded alcohol series. Endogenous peroxidase was suppressed by incubation with 3% hydrogen peroxide for 20 min. For antigen retrieval, the sections were boiled in a pressure cooker containing 10 mM citrate buffer(pH 6.0) for 5 min. The sections were blocked with goat serum in a humid chamber at room temperature for 30 min to reduce non-specific background staining. The sections were further incubated with anti-RhoB antibody at a dilution of 1:100(Rabbit PolyClonal, Proteintech) overnight at 4°C in a moist tray. The sections were also incubated with HRP-conjugated secondary antibody at room temperature for 1h. Afterward, the specimens were washed thrice in PBS, and stained with a liquid DAB substrate kit (ZSGB-BIO, China). Finally, all sections were lightly counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene and mounted. Aa a negative control, duplicate sections were immunostained without the primary antibody. RhoB expression was scored as 0 (negative), 1 (mild, 0% to 4% positive cells), 2 (moderate, 5% to 49% positive cells) and 3 (strong, 50% positive cells)[14].The IHC results were evaluated by two independent pathologists without the knowledge of the patient information.
Plasmid construction and cell transfection The human full-length cDNA fragment of RhoB (GenBank accession no. NM_004040) was obtained using general PCR methods. The construct was sub-cloned into the BamHI / EcoRI site of pcDNA3.0-Flag vector containing an ampicillin resistant gene. pcDNA3.0-Flag-RhoB was then used to over-regulate RhoB expression. The small interfering RNA (siRNA) targeting RhoB was designed with sequence Sense: 5’-ACGUCAUUCUCAUGUGCUUTT-3’; Antisense: 5’-AAGCACAUGAGAAUGACGUTT, for negative control (Sense: 5’-UUCUCCG AACGUGUCACGUTT-3’; anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’). These sequences were designed and synthesized by Shanghai Gene-Pharma Co. (Shanghai, China).
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
The plasmid and siRNA were transfected into A498,786-O and Caki-1 cells using lipo2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The expression levels of RhoB were determined by Western blot analysis.
Cell proliferation assays In brief, after transfection was performed for 48h, approximately 2000 live cells were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 5d. At the time points of 0, 1, 2, 3, 4, 5,and 6 days, 20μL of MTS solution (Cell Titer96 Aqueous, Promega, Madison, WI) was added to the culture wells. After incubation was conducted for 2 h at 37°C, absorbance was recorded at 490 nm by using a microplate reader.
Plate colony formation assay Cells transfected with plasmid and siRNA were seeded at 300 cells per well in six-well plates and incubated at 37°C for two weeks. The cells were fixed with 70% methanol and then stained with crystal violet. The number of positive colonies (more than 50 cells) was counted. All of the experiments were conducted in triplicates.
Flow cytometry analysis of cell cycle and apoptosis The transfected cells were harvested when 70% to 80% confluency was reached. For cell cycle analysis, 2× 105 cells were collected, washed with cold PBS twice and fixed with cold 70% ethanol at 4°C overnight. Afterward, the cells were treated with RNase A for 30 min at 37°C, stained with propidium iodide (PI) solution (BD Biosciences, San Jose, CA) for 30 min at 37°C according to the manufacturer’s protocol and measured by flow cytometry (BD FACS Caton, USA). Cellquest Pro software (BD Biosciences) was used for data acquisition and analysis. For apoptosis analysis, an AnnexinV-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, China) was used according to the manufacturer’s instructions. In brief, 1× 106 cells were obtained and washed twice with PBS. Afterward, the cells were resuspended in FITC-conjugated annexin V binding buffer, incubated with annexinV-FITC/PI in the dark for 10 min and analyzed by flow cytometry using FloMax software.
Wound-healing assay Cells were seeded in a six-well cell culture plate and incubated in medium containing 1% FBS. Confluent monolayer cells were scratched with 200-μl pipette tips. The wells were gently washed twice with the medium to remove detached cells. Pictures were obtained at incubation times of 0 h and 24 h, using a phase contrast microscope with a digital camera. The coverage of scraping area was measured on the basis of viable cells migrating from both sides. The experiments were performed in triplicate
Cell migration and invasion assays Migration and invasion assays were performed using uncoated and Matrigel-coated Transwell (Corning Costar Corp., Cambridge, MA) membrane filter inserts with pore size of 8 μm according to the manufacturer’s instructions. Approximately 2×104 cells per well were suspended in 200 μL medium with 1% FBS and then seeded into the upper chambers filled with 500 μL medium containing 10% FBS. The cells were incubated at 37°C for 12 h and 24h for migration and invasion respectively. The cells on the upper surfaces of the Transwell chambers were removed by cotton swabs;the cells on the lower surface of the membrane were fixed and
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
stained with crystal violet solution. The number of cells was counted in five randomly selected microscope fields.
Statistical analysis The results were represented as the average from triplicate experiments and expressed as mean ±SD. Data were analyzed using the SPSS 18 statistical software package (SPSS Inc, Chicago, IL). Student’ t-test was conducted to compare the different groups. The association between RhoB staining and clinicopathogic parameters of patients with ccRCC was evaluated by using the chi-square test or the Fisher’s exact test. P-value
RhoB Acts as a Tumor Suppressor That Inhibits Malignancy of Clear Cell Renal Cell Carcinoma Weihao Chen1,2☯‡, Shaoxi Niu1☯‡, Xin Ma1, Peng Zhang1, Yu Gao1, Yang Fan1, Haigang Pang1, Huijie Gong1, Donglai Shen1, Liangyou Gu1, Yu Zhang1, Xu Zhang1* 1 The State Key Laboratory of Kidney Diseases, Department of Urology, Military Postgraduate Medical College, Chinese People's Liberation Army General Hospital, Beijing, People's Republic of China, 2 Department of Urology, General Hospital of the Navy, Beijing, China
a11111
☯ These authors contributed equally to this work. ‡ These authors are joint senior authors on this work. * [email protected]
Abstract OPEN ACCESS Citation: Chen W, Niu S, Ma X, Zhang P, Gao Y, Fan Y, et al. (2016) RhoB Acts as a Tumor Suppressor That Inhibits Malignancy of Clear Cell Renal Cell Carcinoma. PLoS ONE 11(7): e0157599. doi:10.1371/journal.pone.0157599 Editor: Hiromu Suzuki, Sapporo Medical University, JAPAN Received: January 23, 2016 Accepted: May 31, 2016 Published: July 6, 2016 Copyright: © 2016 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National High Technology Research and Development Program of China (2014AA020607) to XM and NGH Innovation Fostering Fund (CXPY201423). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
This study aims to investigate the biological role of RhoB in clear cell renal cell carcinoma (ccRCC). The expression of RhoB was examined in specimens of patients and cell lines by Western blot and Immunohistochemistry. The correlation between RhoB expression and clinicopathologic variables was also analyzed. The effects of RhoB on cell proliferation, cell cycle, cell apoptosis, and invasion/migration were detected by over-expression and knockdown of RhoB level in ccRCC cells via plasmids and RNAi. The results showed that RhoB was low-expressed in ccRCC surgical specimens and cell lines compared with adjacent normal renal tissues and normal human renal proximal tubular epithelial cell lines (HKC), and its protein expression level was significantly associated with the tumor pathologic parameter embracing tumor size(P = 0.0157), pT stage(P = 0.0035), TNM stage(P = 0.0024) and Fuhrman tumor grade(P = 0.0008). Further, over-expression of RhoB remarkably inhibited the cancer cell proliferation, colony formation and promoted cancer cell apoptosis, and aslo reduced the invasion and migration ability of ccRCC cells. Interestingly, up-regulation of RhoB could induce cell cycle arrest in G2/M phase and led to cell cycle regulators(CyclineB1,CDK1) and pro-apoptotic protein(casp3,casp9) aberrant expression. Moreover, knockdown of RhoB in HKC cells promoted cell proliferation and migration. Taken together, our study indicates that RhoB expression is decreased in ccRCC carcinogenesis and progression. Up-regulation of RhoB significantly inhibits ccRCC cell malignant phenotype. These findings show that RhoB may play a tumor suppressive role in ccRCC cells, raising its potential value in futural therapeutic target for the patients of ccRCC.
Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
Introduction Clear cell renal cell carcinoma (ccRCC) originates from proximal tubule cells, and is one of the most common histological subtypes of renal cell carcinomas. ccRCC is the second leading cause of death among all types of urologic cancers[1, 2]. In fact, approximately 25% to 30% of the patients with ccRCC present metastasis at the time of diagnosis, and overall survival is usually very poor in the follow-up period[3]. Unfortunately, ccRCC is resistant to conventional cytotoxic agents, in addition to surgery[4].Although the new targeted therapies have produced dramatic clinical effects for the treatment of metastatic renal-cell carcinoma (RCC), such targeted therapies remain unsatisfactory because some patients are resistant to therapy [5].Thus, further studies are necessary to investigate the tumorigenesis and progression of ccRCC and to explore new therapeutic targets to improve the efficiency of ccRCC treatment. RhoB is a member of the Rho family of small GTPases, which regulates actin stress fibers, cytoskeletal actin organization and vesicle transport, in cancer cells, RhoB also modulates proliferation, survival, invasion and angiogenic capacity[6]. Furthermore, RhoB may act as a tumor suppressor in growth control and transformation. RhoB is not mutated in various cancers, but its altered expression and activity are possibly critical to cancer progression and therapeutic responses therapeutic responses[7, 8]. Loss of RhoB expression has been reported in head and neck cancer, lung cancer and gastric cancer[9–11]. RhoB gene knockout in mouse increases the frequency of chemically induced neoplastic transformation[12]. Overexpression of RhoB in human tumor cells results in inhibition of signal transduction pathways involved in oncogenesis and tumor survival, as well as apoptosis[13]. Studies have revealed the putative tumor-suppressive effect of RhoB in human tumor, however, to the best of our knowledge, the function of RhoB in ccRCC remains unclear. In the present study, the relative expression levels of RhoB in ccRCC cell lines and patient specimens were investigated by Western blot and immunohistochemistry. The correlation between RhoB expression and clinicopathological parameters of patients with ccRCC was also analyzed. The biological effects of overexpression and low-expression of RhoB on the malignant phenotypes of ccRCC cell A498,786-O and Caki-1 or normal HKC cells were further examined.
Materials and Methods Ethics Statement All patients authorized the Written Informed Consent. This study was approved by the Protection of Human Subjects Committee, Chinese People’s Liberation Army (PLA) General Hospital.
Cell culture and reagents Human renal proximal tubular epithelial cell line HKC and HK2, and the renal cancer cell lines, including A498, 786-O, 769-P and Caki-1, Caki-2 were preserved in our laboratory. The cells were maintained in DMEM or RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Invitrogen), 100 units/ml of penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C.
Patients and tissue samples All ccRCC cases diagnosed clinically and histopathologically were obtained from Chinese People’s Liberation Army General Hospital (Beijing, China) in 2011. The study were approved by the Chinese People’s Liberation Army General Hospital’s Protection of Human Subjects Committee and the informed consent was obtained from all patients. After resection was performed,
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
specimens were promptly frozen in liquid nitrogen and stored at -80°C until use. In addition, parts of each sample were fixed in formalin, embedded in paraffin and stored in our laboratory. Inclusion criterion included: Patients who received radical nephrectomy in our hospital, the pathologic diagnosis was ccRCC, both the tumor specimens and renal tissues were available after surgery. Exclusion criterion were as follows:patients receiving chemotherapy or radiotherapy before surgery, with multiple renal tumors or distant metastasis.The following clinicopathologic information was collected for each patient,including age, sex, tumor size, pT status and TNM stage.
Protein extraction and Western-blot analysis Whole cell lysate was extracted from each cell line and surgical specimens in RIPA Lysis Buffer (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Protein concentration was measured using BCA Protein Quantitative Kit (Applygen Technologies). Protein (30μg) from each sample was separated in 8% or 12% SDS–PAGE, and transferred to polyvinylidene fluoride membranes (PVDF membranes, Millipore). The PVDF membranes were blocked with 5% low fat milk and then probed with the primary antibody RhoB(1:1000, Proteintech), CyclineB1(1:1000; Immunoway), CDK1(1:1000; Immunoway), cleaved casp9 (1:1000, Epitomics), and cleaved casp3(1:1000, Affinity BioReagents). β-actin(1:1000, ZSGB-BIO) was used as a loading control. Secondary anti-mouse or anti-rabbit antibodies were used at 1:3000 dilutions and the reaction was visualized using an enhanced chemiluminescent Kit (Thermo, IL).
Immunohistochemistry analysis The paraffin sections (4 μm in thickness) were deparaffinized in xylene solvent and rehydrated through a graded alcohol series. Endogenous peroxidase was suppressed by incubation with 3% hydrogen peroxide for 20 min. For antigen retrieval, the sections were boiled in a pressure cooker containing 10 mM citrate buffer(pH 6.0) for 5 min. The sections were blocked with goat serum in a humid chamber at room temperature for 30 min to reduce non-specific background staining. The sections were further incubated with anti-RhoB antibody at a dilution of 1:100(Rabbit PolyClonal, Proteintech) overnight at 4°C in a moist tray. The sections were also incubated with HRP-conjugated secondary antibody at room temperature for 1h. Afterward, the specimens were washed thrice in PBS, and stained with a liquid DAB substrate kit (ZSGB-BIO, China). Finally, all sections were lightly counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene and mounted. Aa a negative control, duplicate sections were immunostained without the primary antibody. RhoB expression was scored as 0 (negative), 1 (mild, 0% to 4% positive cells), 2 (moderate, 5% to 49% positive cells) and 3 (strong, 50% positive cells)[14].The IHC results were evaluated by two independent pathologists without the knowledge of the patient information.
Plasmid construction and cell transfection The human full-length cDNA fragment of RhoB (GenBank accession no. NM_004040) was obtained using general PCR methods. The construct was sub-cloned into the BamHI / EcoRI site of pcDNA3.0-Flag vector containing an ampicillin resistant gene. pcDNA3.0-Flag-RhoB was then used to over-regulate RhoB expression. The small interfering RNA (siRNA) targeting RhoB was designed with sequence Sense: 5’-ACGUCAUUCUCAUGUGCUUTT-3’; Antisense: 5’-AAGCACAUGAGAAUGACGUTT, for negative control (Sense: 5’-UUCUCCG AACGUGUCACGUTT-3’; anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’). These sequences were designed and synthesized by Shanghai Gene-Pharma Co. (Shanghai, China).
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
The plasmid and siRNA were transfected into A498,786-O and Caki-1 cells using lipo2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The expression levels of RhoB were determined by Western blot analysis.
Cell proliferation assays In brief, after transfection was performed for 48h, approximately 2000 live cells were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 5d. At the time points of 0, 1, 2, 3, 4, 5,and 6 days, 20μL of MTS solution (Cell Titer96 Aqueous, Promega, Madison, WI) was added to the culture wells. After incubation was conducted for 2 h at 37°C, absorbance was recorded at 490 nm by using a microplate reader.
Plate colony formation assay Cells transfected with plasmid and siRNA were seeded at 300 cells per well in six-well plates and incubated at 37°C for two weeks. The cells were fixed with 70% methanol and then stained with crystal violet. The number of positive colonies (more than 50 cells) was counted. All of the experiments were conducted in triplicates.
Flow cytometry analysis of cell cycle and apoptosis The transfected cells were harvested when 70% to 80% confluency was reached. For cell cycle analysis, 2× 105 cells were collected, washed with cold PBS twice and fixed with cold 70% ethanol at 4°C overnight. Afterward, the cells were treated with RNase A for 30 min at 37°C, stained with propidium iodide (PI) solution (BD Biosciences, San Jose, CA) for 30 min at 37°C according to the manufacturer’s protocol and measured by flow cytometry (BD FACS Caton, USA). Cellquest Pro software (BD Biosciences) was used for data acquisition and analysis. For apoptosis analysis, an AnnexinV-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, China) was used according to the manufacturer’s instructions. In brief, 1× 106 cells were obtained and washed twice with PBS. Afterward, the cells were resuspended in FITC-conjugated annexin V binding buffer, incubated with annexinV-FITC/PI in the dark for 10 min and analyzed by flow cytometry using FloMax software.
Wound-healing assay Cells were seeded in a six-well cell culture plate and incubated in medium containing 1% FBS. Confluent monolayer cells were scratched with 200-μl pipette tips. The wells were gently washed twice with the medium to remove detached cells. Pictures were obtained at incubation times of 0 h and 24 h, using a phase contrast microscope with a digital camera. The coverage of scraping area was measured on the basis of viable cells migrating from both sides. The experiments were performed in triplicate
Cell migration and invasion assays Migration and invasion assays were performed using uncoated and Matrigel-coated Transwell (Corning Costar Corp., Cambridge, MA) membrane filter inserts with pore size of 8 μm according to the manufacturer’s instructions. Approximately 2×104 cells per well were suspended in 200 μL medium with 1% FBS and then seeded into the upper chambers filled with 500 μL medium containing 10% FBS. The cells were incubated at 37°C for 12 h and 24h for migration and invasion respectively. The cells on the upper surfaces of the Transwell chambers were removed by cotton swabs;the cells on the lower surface of the membrane were fixed and
PLOS ONE | DOI:10.1371/journal.pone.0157599 July 6, 2016
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RhoB and Clear Cell Renal Cell Carcinoma
stained with crystal violet solution. The number of cells was counted in five randomly selected microscope fields.
Statistical analysis The results were represented as the average from triplicate experiments and expressed as mean ±SD. Data were analyzed using the SPSS 18 statistical software package (SPSS Inc, Chicago, IL). Student’ t-test was conducted to compare the different groups. The association between RhoB staining and clinicopathogic parameters of patients with ccRCC was evaluated by using the chi-square test or the Fisher’s exact test. P-value
